Use of at least one substrate of carboxylesterase and/or triacylglycerol lipase for detecting bacteria of the group bacillus cereus

ABSTRACT

Use of at least one chromogenic and/or fluorogenic carboxylesterase and/or triacylglycerol-lipase substrate, to detect bacteria of the  Bacillus cereus  group in a sample capable of containing them.

TECHNICAL FIELD

The present invention relates to the field of microbiological monitoringin the broad sense, such as microbiological monitoring of a sample ofindustrial or clinical origin. More particularly, the present inventiondeals with a method of detecting bacteria belonging to the Bacilluscereus group as distinct from other bacteria, by detecting hydrolysis ofa carboxylesterase and/or triacylglycerol-lipase enzyme substrate.

STATE OF THE ART

The microbiological monitoring of samples of various origins (forexample agri-food or clinical) requires the implementation of techniqueswhich make it possible to detect microorganisms—for example for thepurposes identification and/or enumeration—and of which the yield interms of results must be as fast as possible. Generally, saidmicroorganisms can be non-pathogenic, such as bacteria of technologicalinterest like ferments or quality indicators; the latter make itpossible to validate the production process, from raw materials toprimary products, up to finished products. However, the microorganismswhich are the subject of microbiological monitoring are most frequentlypathogenic, which consequently necessitates their rapid and precisedetection in order to start ad hoc corrective actions as soon aspossible. Of course, the toxins produced by such pathogenicmicroorganisms—and which are wholly or partly responsible for theirpathogenic character—may also be sought.

Clinical diagnosis uses the same techniques: either detection and/orenumeration of the bacteria themselves, or detection of the toxins.

In any case, and whatever the origin of the sample which is the subjectof microbiological monitoring, the important factors for assessing theeffectiveness of this monitoring are: the sensitivity, specificity andtime-to-result.

The genus Bacillus includes Gram-positive bacteria ubiquitously presentin nature: in the earth, water, air, in foodstuffs such as cerealgrains, powdered milk, flour-based products, spices, etc. Their abilityto form spores gives them a great deal of resistance in the outsideenvironment. Spores of Bacillus cereus (B. cereus) may notablycontaminate foodstuffs, from raw materials to manufactured products. Thesurvival of said spores is guaranteed throughout the whole food chain.Under normal circumstances, B. cereus is present in a quantity of lessthan 10³ cells per gram of food and has no pathogenic effect. Thegenerally permissible pathogenic level is of the order of 10⁵ cells pergram of foodstuff. The contamination of an individual from a foodstuffmay therefore be responsible for gastroenteritis. Gastroenteritis linkedto B. cereus manifests itself in either vomiting or diarrhoea. Variousfoodstuffs may be implicated: meat, rice, dried foods, sauces, soups,etc. Opportunistic B. cereus infections may also be observed invulnerable patients such as alcoholic subjects, immunosuppressedsubjects, or following burn-type injuries.

The detection (for example for identification and/or enumerationpurposes) of the bacteria of the Bacillus cereus group is thereforevital, in particular for monitoring laboratories in the agri-foodindustry and for those required to have clinical diagnoses carried out.

As standard, bacteria of the Bacillus cereus group are isolated onconventional selective culture media in dishes: for example, thestandards of the ISO (“International Organization for Standardization”)type or the BAM-FDA (“Bacteriological Analytical Manual of the Food andDrug Administration”) methods recommend using media such as “polymyxinegg yolk mannitol bromothymol blue Agar” (which has the acronym PEMBA)or “mannitol-egg yolk-polymyxin Agar” (which has the acronym MYP).Bacillus cereus is identified in accordance with morphological, culturaland/or metabolic criteria. Unfortunately, these PEMBA or MYP media cangive rise to false-positive results (detection specificity problem) forexample due to a poor inhibiting system, or to false-negative results(sensitivity problem), for example due to the absence, in certainstrains of B. cereus, of key morphological and/or metaboliccharacteristics. Finally, certain strains belonging to the B. cereusgroup exhibit ambiguous reactions, as described by Fricker et al.,International Journal of Food Microbiology; 121 (2008): 27-34.

Chromogenic media in dishes have been developed in order to attempt toreduce/suppress false-negative results. Such chromogenic media containnatural or synthetic chromogenic substrates. Enzyme activities specificto certain bacterial strains are thus detected by the cleaving of thesesubstrates. This has been described numerous times in the state of theart.

Generally, the detection specificity may be improved by adding, into theculture medium, inhibiting systems, antimicrobial and/or antifungalcocktails intended to limit the growth of the microorganisms which arenot targeted/sought. However, such cocktails of inhibitors generallytend to delay the growth of the target/sought microorganisms.

Chromogenic or fluorescent media based on the detection ofPhosphatidylinositol-specific phospholipase C (hereafter named PI-PLC;enzyme classification: EC 4.6.1.13), have been described in particularin patents U.S. Pat. No. 6,284,517, EP-B-1219628 and U.S. Pat. No.6,558,917, as well as in the aforementioned publication by Fricker etal., 2008. Such media present, amongst others, the disadvantage ofgiving rise to false-negative results (detection sensitivity problem),in particular with certain strains of Bacillus cereus, B. mycoides, B.weihenstephanensis which do not exhibit PI-PLC activity, or a low PI-PLCactivity (B. anthracis), or even to false-positives (detectionspecificity problem). Furthermore, the fluorescent substrate 4MU-MIP(4-methylumbelliferyl myo-inositol-1-phosphate) exhibits reducedstability in an aqueous medium, which imposes draconian conditions ofuse, in particular in terms of discontinuous measurement offluorescence, and under precise pH conditions at that, as theaforementioned U.S. Pat. No. 6,558,917 states. More precisely, it isnecessary to perform culturing at acidic pH and then alkalinise themedium to enhance the fluorescence, which is read at the end.

For its part, U.S. Pat. No. 7,309,580, describes a medium in a dishwhich combines a chromogenic substrate of phosphatidylcholine-specificphospholipase C (which has the acronym “PC-PLC”; enzyme classification:EC 3.1.4.3) and a chromogenic substrate of PI-PLC. The respectivecolours of the first substrate and of the second substrate are differentand may also be distinguished from the third colour resulting from anymixing of the products of enzyme reactions of said first and secondsubstrates.

The BCM medium, supplied by Biosynth® AG (Switzerland), uses achromogenic substrate of PI-PLC and a system which inhibits theuntargeted bacterial flora, comprising polymyxin B, trimethoprim,sulfamethoxazole and cycloheximide. The test performance is improvedcompared to standard media, but certain atypical strains may remainincorrectly identified (cf. Fricker et al., 2008, above).

There are chromogenic media based on the hydrolysis of β-glucosidasesubstrates, such as Brilliance™ Bacillus cereus Agar supplied by Oxoid™.However, the use of such chromogenic media generates false-positiveresults with the growth of certain Gram-positive bacteria expressingsuch an enzyme activity despite the presence of an anti-Gram positiveinhibiting system comprising polymyxin B and trimethoprim, as well asfalse negatives (cf. Fricker et al., 2008, above).

The application PCT WO 2011/033224, in the name of the Applicant,discloses a method which is intended to make it possible to detectand/or enumerate bacteria of the Bacillus cereus group with the aid of areaction medium comprising a Gram-negative bacteria inhibitor and afluorescent phosphatidylcholine phospholipase C (PC-PLC) substrate. ThePC-PLC substrate specifically described and exemplified in WO2011/033224, namely 4 MU-CP (4-methyl-umbelliferyl-choline phosphate),is a substrate which makes it necessary to work in liquid medium andwhich proves to be ill-suited—or even unsuited—to detecting and/orenumerating bacteria of the Bacillus cereus group in agar medium.Indeed, after 4 MU-CP is cleaved by PC-PLC, the 4 MU does not remainlocalised in the bacterial colony belonging to the Bacillus cereus groupbut is disseminated widely within the agar medium. The fluorescenceemitted is therefore not only detected around the bacterial colonybelonging to the Bacillus cereus group but also elsewhere. In otherwords, the use of a fluorogenic PC-PLC substrate, such as 4 MU-CP, isill-suited to detecting and/or enumerating bacteria of the Bacilluscereus group in a solid or semi-solid medium, for example in agarmedium. Furthermore, from a general point of view, the fluorogenicPC-PLC substrate which is the subject of WO 2011/033224 necessitates amore or less sophisticated apparatus, such as a UV lamp, in order todetect the fluorescence induced by the cleaving of the fluorogenicsubstrate under the effect of the enzyme activity sought.

PCT application WO 2010/128120 (in the name of Biosynth® AG [CH])describes Aldol®-type signalophores which consist of particular indoxyl(1H-indolyl-3-yl) derivatives, namely indoxyls joined onto the cyclicamine (N-arylated). Amongst the extremely large number of 1H-Indol-3-ylindicators tested in WO 2010/128120 for the purpose of detecting variousbacterial strains, table IV c, presented on pages 79 and 80 of thisdocument, mentions the colorimetric detection of Bacillus cereus strainsby using a C1-Esterase Indicator, which is present at a concentration of0.52 mM in agar medium (“Nutrient Agar”). More precisely, this C1esterase substrate is 1-(2-Benzoylphenyl)-1H-indol-3-yl acetate.

The publication “Yasuo Motoyama et al.: “Rapid and sensitive detectionof viable bacteria in contaminated platelet concentrates using a newlydeveloped bioimaging system”, TRANSFUSION, 1st November 2008” disclosesthe use of a C1 esterase substrate, namely 5(6)-carboxyfluoresceindiacetate (“CFDA”; Invitrogen, Carlsbad, Calif.) to detect severalbacteria species within platelet concentrates, including Bacillus cereusamongst others, by detecting a fluorescence.

However, it emerges that C1 esterase substrates—such as those mentionedin WO 2010/128120 and Yasuo Motoyama et al.—are enzyme substrates withlow specificity and which do not make it possible, in particular, todistinguish bacteria of the Bacillus cereus group from other Bacillusspecies frequently encountered, Bacillus subtilis in particular.

There is therefore a need to develop an application/method which isrelatively simple (namely not requiring the use of a sophisticatedapparatus) and which makes it possible to detect the bacteria of theBacillus cereus group, in a liquid medium and in a semi-solid or solidmedium, not only with very good detection sensitivity but also—and aboveall—with a very good detection specificity, which is sufficient todistinguish the bacteria of the Bacillus cereus group from otherBacillus cereus species frequently encountered, Bacillus subtilis inparticular.

STATEMENT OF THE INVENTION

With regard to the problems mentioned above, the Applicant hassurprisingly discovered that a carboxylesterase and/ortriacylglycerol-lipase substrate of general formula (I) (cf. below)which has an aliphatic hydrocarbon chain X which comprises between 11and 17 carbon atoms notably made it possible:

-   -   to detect the bacteria of the Bacillus cereus group not only in        a liquid medium but also in a semi-solid or solid medium, with a        detection sensitivity and above all a “time-to-result” which is        entirely satisfactory,    -   to distinguish bacteria of the Bacillus cereus group from other        Bacillus cereus species frequently encountered, Bacillus        subtilis in particular, and    -   to avoid having to use sophisticated apparatus; the apparatus        used to implement the aforementioned carboxylesterase and/or        triacylglycerol-lipase substrate of general formula (I) indeed        being relatively simple.

As a consequence, an object of the invention relates to the use of atleast one chromogenic and/or fluorogenic carboxylesterase and/ortriacylglycerol-lipase substrate for detecting the bacteria of theBacillus cereus group in a sample capable of containing them, such as asample of agri-food origin or clinical origin, wherein saidcarboxylesterase and/or triacylglycerol-lipase substrate is a substrateof the general formula (I):

wherein M represents the labelling part, and

X represents an aliphatic hydrocarbon chain,

and wherein said aliphatic hydrocarbon chain comprises a number ofcarbon atoms between 11 and 17, for example between 12 and 16.

Of course, the invention also relates to the embodiment in which aplurality of different enzyme substrates (for example two, three, four,etc.) of general formula (I) are used in combination.

Thus the carboxylesterase and/or triacylglycerol-lipase substrates ofgeneral formula (I) as defined previously make it possible to detect thebacteria of the Bacillus cereus group with an optimal detectionsensitivity, and above all an optimal detection specificity and atime-to-result which is entirely satisfactory, all while using arelatively simple apparatus. In particular, thanks to the optimaldetection specificity obtained by the carboxylesterase and/ortriacylglycerol-lipase substrates according to the invention, it nowproves possible to reliably distinguish the bacteria belonging to theBacillus cereus group from bacteria which are not part of this group. Byway of example, the invention makes it possible to reliably distinguishthe bacteria belonging to the Bacillus cereus group from those belongingto the Bacillus subtilis group.

Additionally, the Applicant has also discovered, against allexpectations, that the use of at least one enzyme substrate of generalformula (I) made it possible to detect the bacteria of the Bacilluscereus group with an optimal detection sensitivity and above all anoptimal detection specificity (making it possible to reliablydistinguish the bacteria belonging to the Bacillus cereus group frombacteria which are not part of this group) even if a lean culture medium(also commonly called a “lean medium” or “lean base”) is used. “Leanculture medium” (or “lean base”) is to be understood according to themeaning commonly accepted in microbiology, namely a culture medium whichcontains a low concentration of nutrients. A reference example of such alean culture medium is the “R2A Agar” medium (European Pharmacopoeia;Cat. No.: 1071).

The aforementioned advantageous techniques are optimal when saidaliphatic hydrocarbon chain X comprises a number of carbon atoms between13 and 15. This therefore represents a preferred embodiment of thepresent invention.

According to a preferred embodiment, the detection of the bacteria ofthe Bacillus cereus group by means of said at least one carboxylesteraseand/or triacylglycerol-lipase substrate of general formula (I) takesplace in a solid or semi-solid medium such as an agar medium.

The above-mentioned carboxylesterase and/or triacylglycerol-lipasesubstrate of general formula (I) is hydrolysed by the carboxylesteraseand/or triacylglycerol-lipase enzymes of the bacteria belonging to theBacillus cereus group according to the following reaction:

X preferably represents an aliphatic hydrocarbon chain, advantageously alinear and saturated aliphatic hydrocarbon chain.

According to one particularly preferred embodiment, the carboxylesteraseand/or triacylglycerol-lipase substrate is chosen from5-bromo-4-chloro-3-indoxyl-myristate, 5-bromo-4-chloro-3-indoxylpalmitate, or a combination of the two.

According to a preferred embodiment, said labelling part M is:

-   -   a chromogenic (chromophore) labelling part selected from:        indoxyls (or at least one of their derivatives, such as Aldols®,        Biosynth AG, Switzerland), alizarin (also called        dihydroxyanthraquinone), hydroxyquinoline, catechol,        dihydroxyflavone, esculetin, nitrophenol, naphthol, or at least        one of their derivatives; advantageously selected from indoxyls,        alizarin, hydroxyquinoline, catechol, dihydroxyflavone,        esculetin, or at least one of their derivatives; or    -   a fluorogenic labelling part (fluorophore) selected from: the        derivatives of fluorescein, of rhodamine, of hydroxyflavone, of        ELF97.

Preferably, M represents a chromogenic labelling part (chromophore),preferably selected from indoxyls, alizarin, hydroxyquinoline, catechol,dihydroxyflavone, esculetin, or at least one of their derivatives.According to a particularly preferred embodiment, said chromogeniclabelling part is based on indoxyl or one of its derivatives.

According to a particularly preferred embodiment, said aliphatichydrocarbon chain X comprises 13 or 15 carbon atoms, preferably 13carbon atoms.

According to a particular embodiment, said carboxylesterase and/ortriacylglycerol-lipase substrate is a substrate based on indoxyl or oneof its derivatives, said carboxylesterase and/or triacylglycerol-lipasesubstrate being used in combination with at least an agent whichpromotes the oxidative polymerisation of the indoxyl derivative, such asa metallic complex of the ammonium ferric citrate type.

With regard to the nomenclature of the enzyme substrates based onindoxyl (or on one of its derivatives), the roots “indoxyl” and“indolyl” should be understood as being equivalent. Thus, X-myristatemay be named either “5-bromo-4-chloro-3-indoxyl-myristate” or“5-bromo-4-chloro-3-indolyl-myristate”.

According to a preferred embodiment, said carboxylesterase and/ortriacylglycerol-lipase substrate is contained in a reaction mediumcomprising at least one, preferably two and advantageously three of thefollowing components:

-   -   a bacterial culture medium suitable for the bacteria to be        detected, preferably a solid or semi-solid medium such as an        agar medium,    -   an anti-Gram negative selective system,    -   an antifungal agent.

Another object of the invention relates to a method for detectingbacteria of the Bacillus cereus group in a solid or semi-solid medium,said method comprising the following steps:

-   -   a) placing a sample capable of containing bacteria of the        Bacillus cereus group in contact, within said solid or        semi-solid medium, with a reaction medium comprising at least        one carboxylesterase and/or triacylglycerol-lipase substrate        such as defined previously and a Gram-negative bacteria        inhibitor, said substrate being a chromogenic and/or fluorogenic        substrate;    -   b) incubating the assembly for a time period sufficient to        enable the appearance of bacterial colonies of the Bacillus        cereus group;    -   c) detecting the bacteria of the Bacillus cereus group through        observing a coloration and/or a fluorescence caused by the        hydrolysis of the carboxylesterase and/or triacylglycerol-lipase        substrate by said bacteria.

Of course, as the chromogenic and/or fluorogenic substrate, a substrateis used which is suitable for use in a solid or semi-solid medium,namely suitable for generating a coloration and/or a fluorescence whichremain(s) localised in the zone of hydrolysis of said substrate by theenzyme activity sought. Indeed, it is important that, unlike substratessuch as 4-MU-CP (4-methyl-umbelliferyl-choline phosphate), thechromogenic and/or fluorogenic substrate according to the inventiongenerates, after hydrolysis, a chromophore and/or fluorophore which isdisseminated very little—or not at all—within the solid or semi-solidmedium but which remains localised on the bacterial colonies of interest(namely on the bacterial colonies formed by the bacteria of the Bacilluscereus group).

The reaction medium preferably comprises a culture medium.

According to a preferred embodiment, said method comprises a prior stepof sample enrichment.

The invention also relates to the use of at least one carboxylesteraseand/or triacylglycerol-lipase substrate as defined above to detect thebacteria of the Bacillus cereus group as distinct from other bacteria.

In the terms of the present invention, the bacteria of the Bacilluscereus group are chosen from Bacillus cereus, Bacillus anthracis,Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides,Bacillus weihenstephanensis, and the other bacteria are chosen fromListeria monocytogenes, Listeria ivanovii, Staphylococcus spp. or theother species of the genus Bacillus spp. such as Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis,Bacillus sphaericus, Bacillus circulans, Bacillus lentus, Bacilluspumilus, Bacillus megaterium.

The invention also relates to a reaction medium for detecting thebacteria of the Bacillus cereus group in a sample capable of containingthem, said reaction medium comprising:

-   -   at least one carboxylesterase and/or triacylglycerol-lipase        substrate such as defined above, and    -   a bacterial culture medium suitable for the bacteria to be        detected, preferably a solid or semi-solid medium such as an        agar medium.

The reaction medium according to the invention preferably furthercomprises at least one anti-Gram negative selective system and/or atleast one antifungal agent.

The present patent application also describes the use of at least onechromogenic and/or flurogenic carboxylesterase and/ortriacylglycerol-lipase substrate for the detection of the bacteria ofthe Bacillus cereus group in a sample capable of containing them, suchas a sample of food origin or clinical origin.

“Sample” is to be understood to be a small part or small isolatedquantity of an entity for analysis. The sample can be of industrialorigin, or, according to a non-exhaustive list, can be an air specimen,a water specimen, a surface specimen, a part or a manufactured product,or a food product. Amongst the samples of food origin, non-exhaustivemention can be made of a sample of dairy products (yogurts, cheeses . .. ), meat, fish, eggs, fruits, vegetables, water, beverages (milk, fruitjuice, soda, etc.). These samples of food origin can also come fromsauces or ready meals. Finally, a food sample can come from an animalfeed, such as notably animal meals. The sample can be of biologicalorigin, either animal, vegetable or human. In this case it maycorrespond to a specimen of biological fluid (in particular whole blood,serum, plasma, urine, cerebrospinal fluid, bronchoalveolar lavage,stools, organic secretion), a tissue specimen or isolated cells. Thisspecimen can be used as-is or, prior to the analysis, undergopreparation by enrichment, extraction, concentration or purification, inaccordance with methods known to the person skilled in the art.

Microbiological monitoring corresponds to the analysis of a sample withthe aim of detecting and/or enumerating microorganisms suspected/capableof being present within said sample. Reaction medium is to be understoodto be a medium comprising all the elements necessary for the survivaland/or for the growth of the microorganisms. This reaction medium mayserve either solely as a revealing medium or as a culture and revealingmedium. In the first case, the culturing of the microorganisms may beperformed before seeding and, in the second case, the reaction mediumalso constitutes the culture medium. The reaction medium may be solid,semi-solid or liquid. A semi-solid or solid medium is understood to be agelled medium, for example. The medium according to the invention ispreferably a gelled medium. Agar is the conventional gelling agent usedin microbiology for culturing microorganisms, but it is possible to useother gelling agents, such as gelrite, gelatine or agarose, for example.A number of agar media are commercially available and known to theperson skilled in the art, such as Columbia agar, Trypcase-soy agar,MacConkey agar, Mueller Hinton agar, for example, or more generallythose described in the Handbook of Microbiological Media (CRC Press).The reaction medium may further comprise one or more elements incombination, such as growth factors such as peptones in particular,carbohydrates, nucleotides, minerals, vitamins, sheep or horse blood,amino acids, salts, buffers, etc. This reaction medium may also compriseat least one dye or pH indicator, which may be a chromophore or afluorophore, preferably a chromophore. As an example of a chromophore,mention can be made of neutral red, aniline blue and bromocresol blue.As a guide, mention can be made of Evans blue, neutral red, phenol red,nitroaniline, malachite green, brilliant green, etc. The medium reactionmay furthermore contain an opacifying agent such as titanium dioxide,kaolin, these being given purely as a guide.

This reaction medium may take the form of a liquid, a ready-to-use gel,i.e. ready to be seeded in a tube, in a flask or on a Petri dish. Withregard to the present invention, the reaction medium additionallycontains a substrate making it possible to detect an enzyme or metabolicactivity of the target microorganisms by means of a directly orindirectly detectable signal.

The detection of the bacteria of the Bacillus cereus group make itpossible to perceive with the naked eye or with the aid of an opticaldevice, the existence of a growth of the target bacteria (in this casebelonging to the Bacillus cereus group), namely the appearance ofcolonies which are coloured and/or fluorescent (depending on whether thesubstrate used is chromogenic, fluorogenic or exhibits bothcharacteristics at the same time), said colonies being coloured and/orfluorescent through the revelation of carboxylesterase-type and/ortriacylglycerol-lipase-type enzyme activity of the bacteria of theBacillus cereus group on said carboxylesterase and/ortriacylglycerol-lipase substrate.

As indicated previously, the detection of the fluorescence emitted aftercleavage of the fluorogenic enzyme substrates makes it necessary to usean optical device, whereas the cleavage of the chromogenic enzymesubstrates may be observed with the naked eye or, if needed, with theaid of an optical device. Advantageously, the detection of the bacteriaof the Bacillus cereus group also makes it possible to identify and/orenumerate them.

The enumeration of the bacteria of the Bacillus cereus group, for itspart, consists in quantifying the number of colonies of bacteria of theBacillus cereus group which has grown on the culture medium by employingmicrobiology techniques well known to the person skilled in the art.

Employing a chromogenic and/or fluorogenic, preferably chromogenic,carboxylesterase and/or triacylglycerol-lipase substrate makes itpossible to obtain highly satisfactory detection sensitivity whileconferring very good specificity: the majority of the Gram-positivebacteria detected are bacteria of the Bacillus cereus group. The otherGram-positive and Gram-negative bacteria are not coloured and/orfluorescent.

Furthermore, such a carboxylesterase and/or triacylglycerol-lipasesubstrate exerts very little inhibition—or even none at all—on thegrowth of the bacteria of the Bacillus cereus group on the culturemedia, which represents a not insignificant advantage.

Chromogenic and/or fluorogenic substrate is to be understood to be asubstrate which makes it possible to detect an enzyme or metabolicactivity of the target/sought microorganisms by means of a directly orindirectly detectable signal. For direct detection, this substrate canbe bound to a part acting as a fluorogenic or chromogenic label (Orengaet al., 2009; J. Microbiol. Methods; 79(2):139-55)), preferablychromogenic in the terms of the present invention. For indirectdetection, the reaction medium according to the invention can alsocontain a pH indicator which is sensitive to the pH variation induced bythe consumption of the substrate and which reveals the metabolism of thetarget microorganisms. Said pH indicator can be a chromophore or afluorophore. As examples of chromophores, mention can be made ofbromocresol purple, bromothymol blue, neutral red, aniline blue andbromocresol blue.

As indicated previously, according to a particularly advantageousembodiment, it is important that the chromogenic and/or fluorogenicsubstrate according to the invention is suitable for use in a solid orsemi-solid medium, namely suitable for generating a coloration and/or afluorescence which remain(s) localised at the zone of hydrolysis of saidsubstrate by the enzyme activity sought. Indeed, it is important that,unlike substrates such as 4 MU-CP (4-methyl-umbelliferyl-cholinephosphate), the chromogenic and/or fluorogenic substrate according tothe invention is disseminated very little—or not at all—within the solidor semi-solid medium but remains localised on the bacterial colonies ofinterest (namely on the bacterial colonies formed by the bacteria of theBacillus cereus group).

“Carboxylesterase and/or triacylglycerol-lipase substrate” should beunderstood to be an enzyme substrate which, after reaction with acarboxylesterase-type enzyme (enzyme classification: E.C. 3.1.1.1)and/or triacylglycerol-lipase-type enzyme (enzyme classification E.C.3.1.1.3), is capable of giving rise to a coloured and/or fluorescentreaction, depending on whether a chromogenic and/or fluorogenicsubstrate is used.

The chromogenic and/or fluorogenic carboxylesterase and/ortriacylglycerol-lipase substrates used according to the presentinvention are preferably synthetic substrates made up of two parts, afirst part which is specific to the enzyme activity to be detected,namely a carboxylesterase-type and/or triacylglycerol-lipase-type enzymeactivity, and a second part which acts as a label, hereafter called the“labelling part”. The labelling part is chromogenic and/or fluorogenic(chromophore and/or fluorophore) when it is no longer associated withthe first part, namely after cleavage by the carboxylesterase and/ortriacylglycerol-lipase enzyme (within the framework of a hydrolysis-typereaction) and separation of said first and second parts.

The chromogenic enzyme substrates (comprising at least one chromophore)usable in the terms of the present invention can be of different kinds.

Firstly, mention should be made of chromophores of the indoxyl type andderivatives thereof which, in the presence of oxygen, produce aprecipitate varying from blue to pink. The addition of an agent whichpromotes oxidative polymerisation of the indoxyl derivative, such as ametal complex of the ammonium ferric citrate type in the culture mediummay prove to be advantageous. The carboxylesterase and/ortriacylglycerol-lipase substrates based on indoxyl and its derivativesare particularly preferred in the terms of the present invention due totheir relatively easy implementation and their good sensitivity fordetecting the target bacteria (notably for the purpose of identifyingand/or enumerating the latter), in this case for detecting the bacteriaof the B. cereus group. The indoxyl-based substrates (by way of example,5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside or X-beta-glucosidecan be cited) are generally well known to the person skilled in the artand are widely used in the chromogenic media on the market. Theirapplications essentially relate to the enzyme activities of the osidase,esterase, lipase and phosphatase types (phosphatase being an esteraseactivity of phosphoric acid). Well suited to a use on a solid orsemi-solid support (filter, agar, electrophoresis gel, etc.), they areless suited to use in liquid medium (formation of a precipitate).

The Aldol®-type chromophores (Biosynth® AG) also represent chromophoresof interest in terms of the present invention, insofar as the appearanceof a coloured precipitate does not require any addition (oxygen, metalsalts, etc.). The use of Aldol®-based enzyme substrates may thereforeprove to be particularly advantageous within the framework of pour plateseeding of bacteria. These Aldol® chromophores are particularderivatives of indoxyl (1H-indolyl-3-yl), namely indoxyls joined ontothe cyclic amine (N-arylated), such as disclosed in the PCT patentapplication published under the reference WO 2010/128120 (in the name ofBiosynth® AG [CH]). Such enzyme substrates can be obtained fromBiosynth® AG.

Secondly, there are chromophores such as hydroxyquinoline,dihydroxyanthraquinone, catechol, dihydroxyflavone or esculetin andtheir derivatives which, in the presence of iron salts, produce acoloured precipitate. Here too, their applications primarily relate toenzyme activities of the osidase, esterase and phosphatase type.

Thirdly, there are naphthol-based enzyme substrates. In this case, theenzyme-substrate reaction is performed in two steps, the naphtholreleased as a result of the expression of enzyme activity undergoes“azo-coupling” in the presence of a diazonium salt which is added at themoment of revelation, leading to the formation of an insoluble colouredcompound. They also make it possible to detect osidase and esteraseactivities via naphthol. The “azo-coupling” reaction is performed in amedium which is often chemically aggressive, toxic to the bacteria andwhich makes the sample unusable for other analyses.

According to a preferred embodiment of the present invention, thelabelling part of the enzyme substrate according to the invention is alabelling part which is preferably chosen from indoxyls and theirderivatives (3-Indoxyl, 5-Bromo-3-indoxyl, 5-Iodo-3-indoxyl,4-Chloro-3-indoxyl, 5-Bromo-4-chloro-3-indoxyl,5-Bromo-6-chloro-3-indoxyl, 6-Bromo-3-indoxyl, 6-Chloro-3-indoxyl,6-Fluoro-3-indoxyl, 5-Bromo-4-chloro-3-indoxyl-N-methyl,N-Methyl-3-indoxyl, Aldol® . . . ); alizarin; hydroxyquinoline;catechol; dihydroxyflavone, hydroxyflavone, naphthol, ELF97, esculetinor one of the derivatives. Preferably, said labelling part is achromogenic labelling part, advantageously based on indoxyl (indolyl) orone of its derivatives.

Preferably, the chromogenic and/or fluorogenic, advantageouslychromogenic, carboxylesterase and/or triacylglycerol-lipase substrateaccording to the present invention is selected from:3-Indoxyl-myristate, 5-Bromo-3-indoxyl-myristate,5-Iodo-3-indoxyl-myristate, 4-Chloro-3-indoxyl-myristate,5-Bromo-4-chloro-3-indoxyl-myristate,5-Bromo-6-chloro-3-indoxyl-myristate, 6-Bromo-3-indoxyl-myristate,6-Chloro-3-indoxyl-myristate, 6-Fluoro-3-indoxyl-myristate,5-Bromo-4-chloro-3-indoxyl-N-methyl-myristate,N-Methyl-3-indoxyl-myristate, Aldol®-myristate; 3-Indoxyl-palmitate,5-Bromo-3-indoxyl-palmitate, 5-Iodo-3-indoxyl-palmitate,4-Chloro-3-indoxyl-palmitate, 5-Bromo-4-chloro-3-indoxyl-palmitate,5-Bromo-6-chloro-3-indoxyl-palmitate, 6-Bromo-3-indoxyl-palmitate,6-Chloro-3-indoxyl-palmitate, 6-Fluoro-3-indoxyl-palmitate,5-Bromo-4-chloro-3-indoxyl-N-methyl-palmitate,N-Methyl-3-indoxyl-palmitate, Aldol®-palmitate, Alizarin-myristate,alizarin-palmitate, 3,4-cyclohexenoesculetin-myristate (CHE-myristate),3,4-cyclohexenoesculetin-palmitate (CHE-palmitate), catechol-myristate,catechol-palmitate, dihydroxyflavone-myristate,dihydroxyflavone-palmitate, hydroxyquinoline-myristate andhydroxyquinoline-palmitate. Advantageously, said substrate is5-bromo-4-chloro-3-indoxyl-myristate (X-C14) or5-bromo-4-chloro-3-indoxyl-palmitate (X-C16). According to a particularembodiment, a combination of 5-bromo-4-chloro-3-indoxyl-myristate(X-C14) and 5-bromo-4-chloro-3-indoxyl-palmitate (X-C16) can be used forthe purposes of the present invention.

According to a particular embodiment, said substrate is acarboxylesterase and triacylglycerol-lipase substrate.

As indicated above, the carboxylesterase and/or triacylglycerol-lipasesubstrate is preferably a chromogenic substrate which can be cleaved bythe carboxylesterase and/or triacylglycerol-lipase activity of bacteriaof the Bacillus cereus group. Thus, this chromogenic substrate is,preferably, a substrate made up of a target part and a labelling part.The hydrolysis of the substrate by the carboxylesterase and/ortriacylglycerol-lipase enzyme of the bacteria of the Bacillus cereusgroup induces the separation (cleavage) of the target part and thelabelling part, said target part characterising the carboxylesteraseand/or triacylglycerol-lipase enzyme activity and said labelling partbeing a molecule which makes it possible to reveal the hydrolysisreaction via the appearance of coloration at the hydrolysis site: on thecolonies.

The person skilled in the art may also use a bi-plate, which makes itpossible to easily compare two media, comprising different substrates ordifferent selective mixtures, onto which the same biological sample willhave been deposited. The reaction medium may comprise one or moreselective agents which make it possible to inhibit the growth of theGram-negative germs, of yeasts and moulds and of Gram-positive bacteriaapart from the bacteria of interest—constituted here by the bacteria ofthe B. cereus group.

“Selective agent” is to be understood to be any compound capable ofpreventing or slowing the growth of a microorganism other than thetarget microorganism. By way of example, antibiotics, antifungals, orlithium chloride may be used. Without being limiting, a concentration ofbetween 0.01 mg/l and 5 g/l of selective agent(s) is particularlysuitable for the present invention.

Concerning the anti-Gram negative (anti-Gram−), anti-Gram positive(anti-Gram+) and/or antifungal selective systems, these latter arewell-known to the person skilled in the art. By way of example, in theanti-Gram negative selective system used for the purposes of the presentinvention, one or more selective agent(s) selected from nalidixic acid,aztreonam, polymyxin B, colistin is/are used at concentrations known tothe person skilled in the art to obtain the desired effect, namely theelimination of the Gram-negative bacteria.

An antifungal (or “antifungal agent”) is understood to be any compoundcapable of preventing or slowing the growth of a yeast or a mould. As aguide, it is possible to mention in particular amphotericin B,fluconazole, itraconazole, voriconazole, cycloheximide and5-fluorocytosine. Preferably, at least one antifungal agent is used atconcentrations known to the person skilled in the art to obtain theaforementioned effect.

An “antibiotic” is understood to be any compound capable of preventingor slowing the growth of a bacterium. In particular, antibiotics belongto the beta-lactam, glycopeptide, aminoglycoside, polypeptide,sulfonamide and quinolone groups. As a guide, it is in particularpossible to mention the antibiotics cefotaxime, cefsulodin, ceftazidime,cefoxitin, ceftriaxone, cefpodoxime, aztreonam, vancomycin, gentamicin,Trimethoprim, nisin, tobramycin, moxalactam, fosfomycin, D-cycloserine,Polymyxin, Colistin, and quinolones such as nalidixic acid.

“Incubate” is to be understood to mean raising to and holding at, forbetween 1 and 48 hours, preferably between 4 and 24 hours, morepreferably between 16 and 24 hours, an appropriate temperature,generally of between 20 and 50° C., preferably between 30 and 40° C.

In the terms of the present invention, the definition of “detectionsensitivity” is identical to the one commonly recognised in the state ofthe art, namely the ability to give a positive result (appearance of acoloured and/or fluorescent reaction) when the target bacterialstrain—in this case belonging to the Bacillus cereus group—is present inthe sample.

The same applies to detection specificity, whose definition conforms tothat recognised in the state of the art, namely as the ability to give anegative result (absence of a coloured and/or fluorescent reaction) whena strain other than the target bacterial strain—in this case belongingto the Bacillus cereus group—is present in the sample tested.

The chromogenic and/or fluorogenic carboxylesterase and/ortriacylglycerol-lipase substrate is used at a concentration sufficientto make it possible to detect the appearance of a coloured and/orfluorescent reaction after cleavage by the bacteria of the Bacilluscereus group. This concentration is known to the person skilled in theart or at the very least is easy for him/her to determine. As anillustration, the concentration of chromogenic and/or fluorogeniccarboxylesterase and/or fluorogenic substrate is generally between 1mg/L and 10 g/L, preferably between 5 mg/L and 6 g/L, preferably between25 mg/L and 2 g/L and advantageously between 25 mg/L and 500 mg/L.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

1.1 Operating Procedure

For the purposes of this example, a lean base is used, the compositionof which is detailed in table 1 presented below:

TABLE 1 composition of the lean base Compounds Concentration in g/LGlucose 0.25 Agar 13 Yeast extract 2 Sodium pyruvate 0.25 NaCl 5 Buffer0.16

The various compounds of this lean base are weighed one by one. Thewhole is taken up in a necessary volume of osmosis-treated water. Themedia are melted at 100° C. and sterilised by autoclaving (121° C. cyclefor 15 minutes).

After the media have returned to 55° C., the5-bromo-4-chloro-3-indoxyl-myristate (X-C₁₄) and5-bromo-4-chloro-3-indoxyl-palmitate (X-C₁₆) enzyme substrates—bothcarboxylesterase and triacylglycerol-lipase substrates—are added. To doso, stock solutions are prepared at 75 g/L in a DMSO-type organicsolvent. A volume of Tween 20 is added to the stock solution volumenecessary to obtain a final concentration of enzyme substrates of 150mg/L.

The media are divided into sterile Petri dishes before being seeded.

Table 2 below summarises the enzyme substrate composition of the mediatested:

TABLE 2 composition of the tested media Lean base Control Concentrationin mg/L medium (T) Medium 1 Medium 2 X-C14 0 150 0 X-C16 0 0 150

39 strains of Bacillus spp were tested on each of the aforementionedthree media. To do so, each of the media was seeded with the aid of a 10μL sterile loop from bacterial suspensions calibrated to 0.5 McF withthe aid of a densitometer; suspensions produced in 0.85% saline(bioMérieux ref. 20040).

The media were then incubated for 48 hours at 37° C. Readings werecarried out after 24 and 48 hours of incubation. The growth (number ofdials and size of the isolated colonies) as well as the colorationintensities obtained (expression of the target enzyme activity) werenoted for each of these two reading times. The table of results belowonly records coloration intensities. However, NG (“no growth”) is notedwhen growth of the strain is inhibited. The intensity is read on a scaleranging from 0 to 4, with 0 for no coloration and 4 for a maximumcoloration intensity.

1.2 Results

The results at 24 and 48 hours of incubation are respectively presentedin tables 3 and 4 below.

TABLE 3 results after 24 hours of incubation Medi- Medi- Medi- StrainsAPI/QI No. um T um 1 um 2 B. cereus 02 04 010 0 1.5 1.5 B. cereus 02 04001 0 1.5 1.5 B. cereus/thuringiensis 611 0 2.5 2.5 B.cereus/thuringiensis 691 0 2 2 B. cereus 01 06 003 0 0 0 B.thuringiensis 02 04 018 0 2.5 2.5 B. thuringiensis 02 04 012 0 2 1.5 B.mycoides 02 04 030 0 0 0 Bacillus weihenstephanensis 09 06 189 0 1 0.5B. cereus/thuringiensis 07 06 021 0 0.5 0.5 B. cereus 11 05 179 0 0 0 B.mycoides 94 05 092 0 1 0.1 B. pseudomycoides 09 02 022 0 1.5 1.5 B.cereus 78 02 085 0 0.5 0 B. subtilis 632 0 0 0 B. licheniformis 574 0 00 B. subtilis ssp spizizenii 08 01 024 0 0 0 B. circulans 02 04 033 0 00 B. circulans 628 0 0 0 B. badius 02 04 032 0 0 0 B. atrophaeus 90 06035 0 0 0 B. amyloliquefaciens 01 02 055 0 0 0 B. licheniformis 514 0 00 B. coagulans 02 04 050 0 0 0 B. pumilus  22 0 0 0 B. lentus 630 0 0 0B. firmus 93 08 075 0 0 0 B. amyloliquefaciens 90 08 024 0 1 0 B. lentus93 08 070 0 0 0 B. pumilus 02 04 036 0 1.5 1.5 B. clausii 98 08 107 0 00 B. vietnamensis 05 05 001 NG NG NG B. subtilis 09 11 166 0 1.5 1 B.megaterium 92 06 025 0 0 0 B. subtilis 91 08 005 0 1.5 1 B.amyloliquefaciens 90 08 027 0 1.5 0 B. licheniformis 88 07 023 0 0 0 B.megaterium 87 05 008 0 0 0 B. firmus 09 07 061 0 0 0 Detectionsensitivity B. cereus group (14) N/A 78.5 71.5 in (%) Detectionspecificity (25) in % N/A 80 88 Key: NG = no growth 0 = growth, nocoloration 1 = low coloration intensity 2 = intermediate colorationintensity 3 = high coloration intensity 4 = maximum coloration intensity

TABLE 4 results after 48 hours of incubation Medi- Medi- Medi- StrainsAPI/QI No. um T um 1 um 2 B. cereus 02 04 010 0 2 2 B. cereus 02 04 0010 2.5 2.5 B. cereus/thuringiensis 611 0 2.5 2.5 B. cereus/thuringiensis691 0 2 2 B. cereus 01 06 003 0 2.5 2.5 B. thuringiensis 02 04 018 0 2.52.5 B. thuringiensis 02 04 012 0 2 2 B. mycoides 02 04 030 0 2 2Bacillus weihenstephanensis 09 06 189 0 2.5 2.5 B. cereus/thuringiensis07 06 021 0 1.5 1.5 B. cereus 11 05 179 0 1 0.5 B. mycoides 94 05 092 02 1.5 B. pseudomycoides 09 02 022 0 2 2 B. cereus 78 02 085 0 1.5 1 B.subtilis 632 0 0 0 B. licheniformis 574 0 0 0 B. subtilis ssp spizizenii08 01 024 0 0 0 B. circulans 02 04 033 0 0 0 B. circulans 628 0 0 0 B.badius 02 04 032 0 0 0 B. atrophaeus 90 06 035 0 0 0 B.amyloliquefaciens 01 02 055 0 0 0 B. licheniformis 514 0 0 0 B.coagulans 02 04 050 0 0 0 B. pumilus  22 0 0 0 B. lentus 630 0 0 0 B.firmus 93 08 075 0 0 0 B. amyloliquefaciens 90 08 024 0 1.5 0.5 B.lentus 93 08 070 0 0 0 B. pumilus 02 04 036 0 2 1.5 B. clausii 98 08 1070 2 1.5 B. vietnamensis 05 05 001 0 0 0 B. subtilis 09 11 166 0 2 1.5 B.megaterium 92 06 025 0 0 0 B. subtilis 91 08 005 0 2 1.5 B.amyloliquefaciens 90 08 027 0 1.5 0 B. licheniformis 88 07 023 0 0 0 B.megaterium 87 05 008 0 0 0 B. firmus 09 07 061 0 0.5 0 Detectionsensitivity B. cereus group (14) N/A 100 100 in (%) Detectionspecificity (25) in % N/A 72 80 Key: NG = no growth 0 = growth, nocoloration 1 = low coloration intensity 2 = intermediate colorationintensity 3 = high coloration intensity 4 = maximum coloration intensity

For the purposes of determining the detection sensitivity andspecificity, a bacterial strain is deemed to be “detected” when theintensity of the coloration revealed is greater than or equal to 0.5when applying the aforementioned scale for determining the colorationintensity.

The detection sensitivity (in %) is defined as follows:

(number of bacteria of the Bacillus cereus group “detected”/total numberof bacteria of the Bacillus cereus group tested)×100.

The detection specificity (in %) is defined as follows:

((total number of non-Bacillus cereus bacteria—number of non-Bacilluscereus bacteria “detected”)/total number of non-Bacillus cereus bacteriatested)×100.

“Non-Bacillus cereus bacteria” is understood to mean bacteria which donot belong to the Bacillus cereus group.

1.3 Conclusion

The use of the carboxylesterase and triacylglycerol-lipase substratesX-C14 and X-C16 (under the implemented conditions) makes it possible todetect the strains of the Bacillus cereus group with a high detectionsensitivity (and also high detection intensities)—especially after 48 hof incubation—while enabling a highly satisfactory detectionspecificity. This is all the more surprising since this detection occursin a “lean base”, a priori less favourable for growth of the bacteria ofthe Bacillus cereus group.

It emerges clearly from this experiment that it is possible todistinguish the Bacillus cereus group from the other Bacillus speciesfrequently encountered, in particular Bacillus subtilis, by means of thesubstrates X-C₁₄ and X-C₁₆.

EXAMPLE 2

2.1 Operating Procedure

In the autoclaved lean base in example 1 (the composition of which ispresented in the aforementioned table 1), brought back to 55° C.,different chromogenic or fluorogenic substrates were tested withdifferent surface active agents (surfactants). 31 bacterial strains (ofthe genus Bacillus) were spot-test seeded, at 1 μL apiece from 0.5MacFarland suspensions calibrated with the aid of a densitometer. Thedishes were then incubated aerobically, for 24 hours, at 30-35° C.

The chromogenic enzyme substrates 5-bromo-4-chloro-3-indoxyl-myristate(X-C₁₄) and 5-bromo-4-chloro-3-indoxyl-palmitate (X-C₁₆) were used so asto obtain final enzyme substrate concentrations of 0.15 g/L, startingfrom 50 g/L stock solutions.

The following surfactants were used:

-   -   Tween 20 6 g/L, and    -   Triton X305: 3 and 6 g/L

2.2 Results

The results thus obtained are presented below, in Table 5.

TABLE 5 Results Fertility of Fertility of strains of the Sensitivitystrains of the Fertility of B. cereus of B. cereus B. subtilis otherSpecificity group group group Bacillus sp (in %) Media (17 strains) in(%) (5 strains) (9 strains) (14 strains) Lean base (growth control)100%  N/A N/A 78% N/A Lean base + MPLD + TWEEN 94% 78% 20 Lean base +MPLD + Triton 82% 78% X305 3 g/L Lean base + MPLD + Triton 82% 78% X3056 g/L X-C14/Tween 20 6 g/L 94%  88% 100% 78% 93% X-C14/Triton X305 3 g/L88% 100% 100% 89% 86% X-C14/Triton X305 6 g/L 82% 100% 100% 78% 93%X-C16/Triton X305 3 g/L 88% 100% 100% 89% 93% X-C16/Triton X305 6 g/L94% 100% 100% 56% 93% MPLD: 1-methyl-2-pyrrolidinone

The use of an indoxyl-based substrate containing 14 or 16 carbon atomsmakes it possible to obtain an optimal distinction between the bacteriaof the B. cereus group and those which do not belong to it, inparticular between the two main groups which are B. cereus and B.subtilis.

2.3 Conclusion:

A distinction is possible between the bacteria of the B. cereus groupand those which do not belong to this—in particular between the 2 maingroups which are B. cereus and B. subtilis—with the fluorogenic orchromogenic enzyme substrates used. This distinction proves to beoptimal when the enzyme substrate used is an indoxyl-based substratecontaining 14 or 16 carbon atoms.

EXAMPLE 3

3.1 Operating Procedure

In the autoclaved lean base in example 1 (the composition of which ispresented in the aforementioned table 1), brought back to a temperatureof 55° C., substrates of esterases (carboxylesterase) based on alizarinwith carbon chains ranging from 12 to 16 carbon atoms were tested. Thestock solutions of the substrates were produced at concentrations of12.5, 25 and 50 g/L solubilised in DMSO-type organic solvent, mixed witha Tween-type surfactant and added to the media such that the endconcentration of substrates was respectively 50, 100 and 200 mg/L,always with same quantity of solvent and surfactant added to the finalmedia.

The bacterial strains were spot-test seeded, at 1 μL apiece from 0.5MacFarland suspensions calibrated with the aid of a densitometer. Thedishes were then incubated aerobically, for 24 hours, at 30-35° C.

3.2 Results

The results thus obtained are presented below, in table 6.

TABLE 6 Results Fertility of Sensitivity Fertility of Fertility of B.cereus of B. cereus B. subtilis other Specificity group group groupBacillus sp (in %) Media (17 strains) in (%) (5 strains) (9 strains) (14strains) Lean base growth control 100%  N/A N/A 78% N/A Lean base +MPLD + Tween 94% 78% 20 Alizarin-C12/Tween 20 88% 88% 100% 67% 64% 6 g/LAlizarin-C14/Tween 20 88% 94% 100% 78% 79% 6 g/L Alizarin-C16/Tween 2094% 100%  100% 78% 64% 6 g/L

For the purposes of determining the detection sensitivity andspecificity, a bacterial strain is deemed to be “detected” when thecoloration intensity revealed is greater than or equal to 0.5 whenapplying the scale for determining the coloration intensity which ispresented in point 1.2 above.

The detection sensitivity and specificity are also determined asdetailed in point 1.2 above.

3.3 Conclusion

The alizarin-based substrates make it possible to distinguish thebacteria of the B. cereus group from bacteria which do not belong tothis group with very good sensitivity and satisfactory specificity(greater than 60%), the greatest specificity being achieved with the“Alizarin-C14” enzyme substrate, containing 14 carbon atoms.

1. Use of at least one chromogenic and/or fluorogenic carboxylesteraseand/or triacylglycerol-lipase substrate to detect the bacteria of theBacillus cereus group in a sample capable of containing them, such as asample of agri-food origin or clinical origin, wherein saidcarboxylesterase and/or triacylglycerol-lipase substrate is a substrateof the general formula (I):

wherein M represents the labelling part, and X represents an aliphatichydrocarbon chain, and wherein said aliphatic hydrocarbon chaincomprises a number of carbon atoms between 11 and
 17. 2. The useaccording to claim 1, wherein X represents a linear aliphatichydrocarbon chain.
 3. The use according to claim 1 or 2, wherein Xrepresents a linear and saturated aliphatic hydrocarbon chain.
 4. Theuse according to one of claims 1 to 3, wherein the labelling part M is:a chromogenic labelling part selected from: indoxyls, alizarin,hydroxyquinoline, catechol, dihydroxyflavone, esculetin, nitrophenol,naphthol, or at least one of their derivatives; advantageously selectedfrom indoxyls, alizarin, hydroxyquinoline, catechol, dihydroxyflavone,esculetin, or at least one of their derivatives; or a fluorogeniclabelling part selected from: the derivatives of fluorescein, ofrhodamine, of hydroxyflavone, of ELF97.
 5. The use according to one ofclaims 1 to 4, wherein M represents a chromogenic labelling part,preferably selected from indoxyls, alizarin, hydroxyquinoline, catechol,dihydroxyflavone, esculetin, or at least one of their derivatives;advantageously based on indoxyl or one of its derivatives.
 6. The useaccording to one of claims 1 to 5, wherein said aliphatic hydrocarbonchain X comprises a number of carbon atoms between 13 and
 15. 7. The useaccording to one of claims 1 to 6, wherein said aliphatic hydrocarbonchain X comprises 13 or 15 carbon atoms, preferably 13 carbon atoms. 8.The use according to one of claims 1 to 7, wherein said carboxylesteraseand/or triacylglycerol-lipase substrate is a substrate based on indoxylor one of its derivatives, said carboxylesterase and/ortriacylglycerol-lipase substrate being used in combination with at leastan agent which promotes the oxidative polymerisation of the indoxylderivative, such as a metal complex of the ammonium ferric citrate type.9. The use according to one of claims 1 to 8, wherein saidcarboxylesterase and/or triacylglycerol-lipase substrate is contained ina reaction medium comprising at least one, preferably two andadvantageously three of the following components: a bacterial culturemedium suitable for the bacteria to be detected, preferably a solid orsemi-solid medium such as an agar medium. an anti-Gram negativeselective system, an antifungal agent.
 10. A method for detectingbacteria of the Bacillus cereus group in a solid or semi-solid mediumsuch as an agar medium, said method comprising the following steps: a)placing a sample capable of containing bacteria of the Bacillus cereusgroup in contact, within said solid or semi-solid medium, with areaction medium comprising at least one carboxylesterase and/ortriacylglycerol-lipase substrate such as defined in one of claims 1 to 8and a Gram-negative bacteria inhibitor; b) incubating the assembly for atime period sufficient to enable the appearance of bacterial colonies ofthe Bacillus cereus group; c) detecting the bacteria of the Bacilluscereus group through observing a coloration and/or a fluorescence causedby the hydrolysis of the carboxylesterase and/or triacylglycerol-lipasesubstrate by said bacteria.
 11. The method according to claim 10,wherein the reaction medium comprises a culture medium.
 12. The methodaccording to claim 10 or 11, said method comprising a prior step ofsample enrichment.
 13. The use according to one of claims 1 to 9, or themethod according to one of claims 10 to 12, to detect the bacteria ofthe Bacillus cereus group as distinct from other bacteria, said bacteriaof the Bacillus cereus group being chosen from Bacillus cereus, Bacillusanthracis, Bacillus thuringiensis, Bacillus mycoides, Bacilluspseudomycoides, Bacillus weihenstephanensis, and the other bacteriabeing chosen from Listeria monocytogenes, Listeria ivanovii,Staphylococcus spp. or the other species of the genus Bacillus spp. suchas Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus atrophaeus,Bacillus licheniformis, Bacillus sphaericus, Bacillus circulans,Bacillus lentus, Bacillus pumilus, Bacillus megaterium.
 14. A reactionmedium for detecting bacteria of the Bacillus cereus group in a samplecapable of containing them, said reaction medium comprising: at leastone carboxylesterase and/or triacylglycerol-lipase substrate such asdefined in one of claims 1 to 8, and a bacterial culture medium suitablefor the bacteria to be detected, preferably a solid or semi-solid mediumsuch as an agar medium.
 15. The reaction medium according to claim 14,said reaction medium further comprising at least one anti-Gram negativeselective system and/or at least one antifungal agent.